Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, 184-8588, Japan.
Drug Discovery Initiative, The University of Tokyo, Tokyo, 113-0033, Japan.
Biochem Biophys Res Commun. 2021 Jan 22;537:57-63. doi: 10.1016/j.bbrc.2020.12.046. Epub 2020 Dec 29.
Abscisic acid (ABA) is a major phytohormone that regulates abiotic stress responses and development. SNF1-rerated protein kinase 2 (SnRK2) is a key regulator of ABA signaling. To isolate compounds which directly affect SnRK2 activity, we optimized a fluorescence-based system for high-throughput screening (HTS) of SnRK2 kinase regulators. Using this system, we screened a chemical library consisting of 16,000 compounds and identified ten compounds (INH1-10) as potential SnRK2 inhibitors. Further characterization of these compounds by in vitro phosphorylation assays confirmed that three of the ten compounds were SnRK2-specific kinase inhibitors. In contrast, seven of ten compounds inhibited ABA-responsive gene expression in Arabidopsis cells. From these results, INH1 was identified as a SnRK2-specific inhibitor in vitro and in vivo. We propose that INH1 could be a lead compound of chemical tools for studying ABA responses in various plant species.
脱落酸（ABA）是一种主要的植物激素，调节非生物胁迫反应和发育。SNF1 相关蛋白激酶 2（SnRK2）是 ABA 信号转导的关键调节剂。为了分离直接影响 SnRK2 活性的化合物，我们优化了基于荧光的高通量筛选（HTS）SnRK2 激酶调节剂的系统。使用该系统，我们筛选了由 16000 种化合物组成的化学文库，并鉴定出 10 种化合物（INH1-10）为潜在的 SnRK2 抑制剂。通过体外磷酸化实验进一步表征这些化合物证实，这 10 种化合物中有 3 种是 SnRK2 特异性激酶抑制剂。相比之下，10 种化合物中有 7 种抑制拟南芥细胞中 ABA 应答基因的表达。基于这些结果，INH1 被鉴定为体外和体内 SnRK2 特异性抑制剂。我们提出，INH1 可能是研究各种植物物种中 ABA 反应的化学工具的先导化合物。
